TY - JOUR
AU - Burmeister, David M
AB - Abstract Severe thermal injury induces metabolic and physiological stress, prompting a disruption in the hypothalamic-pituitary-adrenal axis. The objective of this study was to evaluate potential confounding effects of Lactated Ringer’s (LR) resuscitation on adrenal damage and cortisol production following burn. Anesthetized swine were instrumented with jugular catheters and sustained 40% TBSA burns from brass probes heated to 100°C. Animals recovered to consciousness and received IV fluid resuscitation with LR at two different volumes: 15 ml/kg/d (limited volume [LV], n = 6) or 2 ml/kg/%TBSA/d (modified Brooke [MB], n = 6). Nonburned animals (Sham) were both oral and IV fluid restricted (S-FR, n = 4) to induce stress. Computed tomography (CT) angiographies were performed at baseline (BL) and 48 hours postburn, while blood and urine samples were collected at BL, 6, 24, and 48 hours postburn, with euthanasia at 48 hours for adrenal harvesting. Urinary cortisol was elevated following burn/surgery in all animals and returned back to BL in S-FR (404 ± 48 pg/mg creatinine) but not MB (1332 ± 176 pg/mg creatinine; P = .005) or LV (1223 ± 335 pg/mg creatinine; P = .07) by 48 hours. Gene expression of cleavage enzymes (3β-HSD, CYP17, CYP11, and CYP21) along the cortisol synthesis pathway showed minimal changes. Adrenal apoptosis (Terminal deoxynucleotidyl transferase dUTP nick-end labeling [TUNEL] staining) was greatest in the MB group (P ≤ .01) when compared to S-FR, partly due to elevations in c-Jun N-terminal kinase. Adrenal hemorrhaging was also greatest in MB animals, with no differences in tissue volume or wet-to-dry ratio. However, tissue levels of cytokines IL-1β, IL-10, and IL-12 were greatest in LV. Burn injury elevates urinary cortisol and compromises adrenal gland integrity, which is affected by IV fluid volume. Severe burns produce pathological changes that result in hemodynamic fluctuations, reduced organ perfusion, hypermetabolism, and a profound inflammatory response, all of which are known precursors to multiple organ dysfunction.1 These systemic disturbances also initiate a stress response that lasts from the time of injury until well after wounds are healed. Acute fluid resuscitation is critical for survival and outcomes after burn injury, with the overall goal of maintaining adequate tissue perfusion. Currently, consensus guidelines set by the American Burn Association recommend lactated Ringer’s solution (LR) at an initial rate of 2 to 4 ml/kg/% TBSA in the first 24 hours for intravenous (IV) fluid of burn injury. If not carefully titrated to urine output thereafter, excessive resuscitation volumes can cause edema/fluid overload, compartment syndromes, and acute respiratory distress syndrome2,3 further exacerbating stress. On the contrary, if patients are under-resuscitated, organ perfusion is not adequately maintained which leads to significant morbidity and mortality. The hypothalamic-pituitary-adrenal (HPA) axis is responsible for the synthesis and secretion of glucocorticoids (ie, cortisol) whose metabolic effects are essential for adaptation to stress. The concentrations of plasma cortisol are elevated proportionally to the %TBSA burned.4–6 The body’s response to burn stimuli coupled with emotional stress in conscious patients alters adrenocortical activity, further delaying healing. Chronic elevation of glucocorticoids is known to suppress the immune system and increase susceptibility to disease.7 The role of HPA axis in burn is characterized by dramatic alterations in cortisol levels, which may last months to years after patients survive the initial burn injury.8,9 Case reports, autopsy studies, and clinical investigations in patients with burns have demonstrated that damage and/or dysfunction of the adrenal glands (eg, hemorrhage, insufficiency) negatively impacts patient outcome.10–15 The incidence of adrenal hemorrhage in patients who succumbed to their burn injury was reported as 27.5% according to macroscopic observation during autopsy.10 Upon microscopic examination, most notable was the evidence of congested blood vessels within the adrenals of burn patients.11 The prolonged elevation in cortisol secretion coupled with hemodynamic fluctuations challenge adrenal glands, and can be associated with adrenal hemorrhaging. Computed tomography (CT), ultrasound, magnetic resonance imaging, and autopsy findings are reported tools for diagnosing adrenal hemorrhage. Of the quarter of burn patients with adrenal hemorrhage, there is a higher prevalence in males.10 Although rare, acute adrenal insufficiency in patients with burns has been observed with rapid onset and sudden deterioration of the patient.15 Dysfunction of the HPA axis in response to other critical illnesses has recently been coined Critical Illness-Related Corticosteroid Insufficiency (CIRCI) in 2008. While adrenal insufficiency and its incidence in critically ill patients has been recognized for some time (first described in 1855)14 specific mechanistic research in burn injury is lacking. Additionally, administration of hormones and hormone mimetics (eg, oxandrolone), following burn has generated recent interest and proven beneficial in some patients.16 This may render the physiological role of the HPA axis even more important. In rodent models, glucocorticoid receptor antagonist treatment has been shown to abolish burn-induced muscle proteolysis17 and expression of myostatin mRNA,18 as well as reduce apoptosis in the thymus and spleen.19 Since intact adrenal signaling is essential for patient outcome following burn injury, we aimed to investigate acute changes in burn-induced adrenal pathophysiology over the first 48 hours after injury. Furthermore, to identify if severity of damage is altered by resuscitation volumes, two different levels of IV LR was given at a low (15 ml/kg/d) and large (Modified Brooke Formula) doses. We hypothesized that IV LR fluids dose-dependently alter adrenal response after burn injury. METHODS Animals Sixteen female Yorkshire swine weighing 41.4 ± 0.6 kg, free of parasites, and infection were included in this study. Animals had a minimum seven day acclimation period during which they were singly housed with ad libitum access to water, and fed a commercial laboratory pelleted diet formulated for pigs. Research was conducted in compliance with the Animal Welfare Act, the implementing Animal Welfare Regulations, and the principles of the Guide for the Care and Use of Laboratory Animals, National Research Council. The facility’s Institutional Animal Care and Use Committee approved all research conducted in this study. The facility where this research was conducted is fully accredited by AAALAC International. Thermal Injury Following the acclimation period, animals were fasted overnight and anesthetized with an intra-muscular injection of tiletamine-zolazepam (Telazol, 6 mg/kg), intubated, and placed on a ventilator with an initial tidal volume at 10 ml/kg, a peak inspiratory pressure at 20 cm H2O, and respiration rate of 8 to 10 breaths/min. End tidal PCO2 of 40 ± 5 mm Hg was maintained on the ventilator and 1 to 3% isoflurane. Creation of the burn wounds and postoperation animal care were performed as previously described.20,21 Briefly, hair was removed from the dorsum, flanks, and legs using clippers and razors with shaving cream. For the sampling of blood and administration of IV fluids, standard cut-down procedures were used to place left and right jugular vein catheters, which were anchored in place and tunneled subcutaneously to the back of the neck. All animals (including shams) were given a one-time (0.1–0.24 mg/kg) intramuscular injection of Buprenex-HCl Sustained Release (Veterinary Technologies/ZooPharm, Windsor, CO) for analgesia which the manufacturers have shown to be bioavailable in large animals for 72 hours. Large (9 × 15 cm) and small (5 × 5 cm) custom designed brass blocks equipped with a thermocouple were maintained at 100 ± 0.2°C by a temperature controller. Heated probes were placed against the skin for 30 seconds to produce full thickness burn injuries as previously described,22 which was repeated until 40% of the TBSA was burned22,23 (Supplementary Figure 1). Burn wounds were covered with Ioban antimicrobial dressings (3M, St. Paul, MN) for the duration of the experiment and replaced if wounds were exposed. All animals were monitored constantly during the day through interaction and remote vivarium camera access to monitor health and behavior. Animal Treatment Groups During the experimental treatment, all animals had unlimited access to the dry pelleted pig diet, while only burned animals were given 15 ml/kg/d of drinking fluids. Animals were randomly assigned to one of three study groups. The first group was IV fluid resuscitation with LR at a limited volume (LV) of 15 ml/kg/d (n = 6). The second group received a larger IV volume of LR that was calculated according to the predicted standard of care modified brooke (MB) formula (2 ml/kg/%TBSA/d) (n = 6). Nonburned animals (Sham) underwent the same procedures except with room temperature blocks, but were deprived access to both oral and IV fluids (S-FR) to induce some level of stress. Animals recovered to full consciousness and were kept in a metabolic cage for separation of urine and feces, which also allowed for control of administering IV and oral fluids according to the treatment group. Resuscitation was performed through the indwelling jugular vein catheter via an infusion pump primed with sterile LR warmed to 37°C according to their body weight the morning of study. For the LV group, the entire volume was infused within 15 minutes, with the same volumes given on day 1 and day 2. For the MB group, on the first day half of the LR was administered in the first 8 hours and the other half in the next 16 according to standard care practice. For the MB group on the second day, while the total volume given was the same, the rate was set constant throughout the course of the day. If animals showed signs of distress (eg, vocalization, jumping, aggression) they were administered Midazolam (0.1–0.25 mg/kg) IM. Twenty-four and forty-eight hours following creation of the burn injuries, animals were mildly sedated with Telazol (6 mg/kg) to collect blood samples and record physiological parameters such as heart rate, respiratory rate, and rectal temperature. Heart rate and respiratory rate were measured manually by a veterinary technician by counting the number of heart beats and breaths for 15 seconds, and then multiplying by 4. CT Angiography At baseline (BL) and 48 hours, contrast-enhanced angiographies were performed under anesthesia. Animals were positioned prone, and 40 ml contrast agent (Isovue-370; Iopamidol 755 mg/ml; contains sodium 0.053 mg, organically bound iodine 370 mg/ml) was injected via an ear vein catheter and CT angiographies were initiated. The left adrenal was reconstructed for quantification of volume and Hounsfield units (HU) using VitreaAdvanced Version 6.7.4 (Vital Image Inc., Minnetonka, MN). After CT scanning, euthanasia was performed with 10 ml of Fatal Plus (Vortech Pharmaceuticals, Dearborn, MI), and adrenal glands were either snap frozen in liquid nitrogen with storage at −80°C, or preserved in 10% neutral buffered formalin, with a ~1 g subsection saved for wet-to-dry weight analysis. Histology Adrenals were preserved in 10% neutral buffered formalin for a minimum of 48 hours, embedded in paraffin wax, and sectioned into 4 µm slices. Hematoxylin and Eosin (H&E) staining was performed according to the manufacturer’s instructions (Sigma Life Science, St. Louis, MI). Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) and 4’,6-diamidino-2-phenylindole (DAPI) stains were performed according to the manufacturer’s protocol. Whole adrenal slices were imaged using an AxioScanZ1 slide scanner (Carl Zeiss, Thornwood, NY). Adrenal sections were put through automated quantification of colors with ImageJ software version 1.51d (Bethesda, MD). This software is equipped with a specific H&E color deconvolution tool that separates colors into channels. The pink/red channel containing red blood cells was set to a threshold of 90 and 255 allowing for an objective quantification of hemorrhaging throughout the entire cross-section of the adrenal. Three regions of interest within the adrenal to include: the glomerulosa, fasiculata/reticularis, and medulla were outlined using the region of interest tool. Within each region of interest within the adrenal cross-section the % surface area value of the pink/red channel was used as an indicator of red blood cell abundance (ie, hemorrhaging). For the analysis of TUNEL staining, four high-magnification images were taken from both the adrenal cortex and the adrenal medulla for each animal. Ensuing images were separated into red, green, and blue channels for the quantification of channel intensities, with apoptosis reported as the mean intensity of the green channel. Blood and Urine Analysis At BL and hours 6, 12, 24, and 48, urine samples were collected into 50 ml tubes, and blood samples were collected into K2 EDTA containing tubes and centrifuged at 4300×g for 10 minutes. Plasma was aliquoted, and stored at −80°C until analysis. Cortisol kit was purchased from Cayman (Ann Arbor, MI) and performed according to the manufacturers’ protocol for plasma and urine. Blood samples were also collected into a lithium heparin containing tube and centrifuged at 4300×g. Serum glucose and cholesterol, as well as urine creatinine were analyzed on a Siemens Dimension Xp and Plus Clinical Chemistry System. Real-time Polymerase Chain Reaction and Protein Quantification RNA was isolated from snap-frozen adrenal glands using Trizol (Sigma-Aldrich, St. Louis, MO) according to the manufacturer’s protocol. RNA quantity was obtained using Nanodrop ND 1000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE) at 260 nm and 280 nm. Genomic DNA removal and first-strand cDNA synthesis was performed according to the RT2 First Strand Kit from (Qiagen, Hilden, Germany). Prepared cDNA samples were diluted in SYBR (Bio-Rad Laboratories, Hercules, CA) with previously published primers24–26 and analyzed in duplicate using a i-Q5 real-time polymerase chain reaction detection system. Sequence specificity was completed using a melting curve with a 0.5°C temperature decrease from 55 to 95°C. Reference genes used to standardize variability include cyclophilin A (Ppia) and glyceraldehyde-3-phosphate dehydrogenase (Gapdh). For protein cytokine analysis, a porcine-specific multiplex kit (EMD Millipore, Billerica, MA) was used according to the manufacturer’s instructions. Statistical Analysis GraphPad Prism was used for graphical representation of data and statistical analysis. Data with repeated measures and histological analysis within the glomerulosa, fasiculata/reticularis, and medulla of the adrenal gland were analyzed using two-way analysis of variance method. Adrenal volume and perfusion were analyzed using a one-way analysis of variance. Protein levels, and wet-to-dry ratios were analyzed using unpaired t test. All data are presented as mean ± SEM. RESULTS Burn Injury Elevates Temperature One animal from the S-FR group did not tolerate the metabolic cage (alertness to the point of excessive vocalization, jumping, etc.) and was therefore removed from the study. Table 1 shows changes in rectal temperature, heart rate, and respiratory rate at BL, 24, and 48 hours following burn injury. At 48 hours, temperature was elevated (P = .005) in LV (39.7 ± 0.3°C) and MB (39.7 ± 0.03°C) when compared with the S-FR animals (38.5 ± 0.2°C). Heart and respiratory rate were similar at BL and postinjury in all animals regardless of treatment. Table 1. Temperature (°C), heart rate (beats/min), respiratory rate (breaths/min), and glucose (mg/dl) were collected at BL, 24 and 48 h after burn injury . Temperature . Heart Rate . Respiratory Rate . Glucose . Timepoint . S-FR . LV . MB . S-FR . LV . MB . S-FR . LV . MB . S-FR . LV . MB . BL 38.5 ± 0.2 38.6 ± 0.2 38.4 ± 0.3 148 ± 22 126 ± 12 135 ± 5 33 ± 3 46 ± 4 43 ± 3 77.3 ± 8.2 57.5 ± 7.9 77.2 ± 5.2 24 h 38.8 ± 0.1 39.3 ± 0.2 38.7 ± 0.2 136 ± 11 136 ± 15 120 ± 10 52 ± 11 43 ± 6 46 ± 10 192.3 ± 38.2 191.0* ± 25.3 159.5 ± 16.8 48 h 38.5 ± 0.2 39.7* ± 0.3 39.7* ± 0.3 128 ± 14 138 ± 14 141 ± 8 38 ± 8 41 ± 5 33 ± 1 196.0 ± 28.7 240.3* ± 59.8 130.7 ± 22.3 . Temperature . Heart Rate . Respiratory Rate . Glucose . Timepoint . S-FR . LV . MB . S-FR . LV . MB . S-FR . LV . MB . S-FR . LV . MB . BL 38.5 ± 0.2 38.6 ± 0.2 38.4 ± 0.3 148 ± 22 126 ± 12 135 ± 5 33 ± 3 46 ± 4 43 ± 3 77.3 ± 8.2 57.5 ± 7.9 77.2 ± 5.2 24 h 38.8 ± 0.1 39.3 ± 0.2 38.7 ± 0.2 136 ± 11 136 ± 15 120 ± 10 52 ± 11 43 ± 6 46 ± 10 192.3 ± 38.2 191.0* ± 25.3 159.5 ± 16.8 48 h 38.5 ± 0.2 39.7* ± 0.3 39.7* ± 0.3 128 ± 14 138 ± 14 141 ± 8 38 ± 8 41 ± 5 33 ± 1 196.0 ± 28.7 240.3* ± 59.8 130.7 ± 22.3 Values are presented as mean ± SEM and * indicates a significant (P < .05) difference from the BL value. S-FR (n = 3), LV (n = 6), MB (n = 6). BL, baseline; LV, limited volume; MB, modified brooke. Open in new tab Table 1. Temperature (°C), heart rate (beats/min), respiratory rate (breaths/min), and glucose (mg/dl) were collected at BL, 24 and 48 h after burn injury . Temperature . Heart Rate . Respiratory Rate . Glucose . Timepoint . S-FR . LV . MB . S-FR . LV . MB . S-FR . LV . MB . S-FR . LV . MB . BL 38.5 ± 0.2 38.6 ± 0.2 38.4 ± 0.3 148 ± 22 126 ± 12 135 ± 5 33 ± 3 46 ± 4 43 ± 3 77.3 ± 8.2 57.5 ± 7.9 77.2 ± 5.2 24 h 38.8 ± 0.1 39.3 ± 0.2 38.7 ± 0.2 136 ± 11 136 ± 15 120 ± 10 52 ± 11 43 ± 6 46 ± 10 192.3 ± 38.2 191.0* ± 25.3 159.5 ± 16.8 48 h 38.5 ± 0.2 39.7* ± 0.3 39.7* ± 0.3 128 ± 14 138 ± 14 141 ± 8 38 ± 8 41 ± 5 33 ± 1 196.0 ± 28.7 240.3* ± 59.8 130.7 ± 22.3 . Temperature . Heart Rate . Respiratory Rate . Glucose . Timepoint . S-FR . LV . MB . S-FR . LV . MB . S-FR . LV . MB . S-FR . LV . MB . BL 38.5 ± 0.2 38.6 ± 0.2 38.4 ± 0.3 148 ± 22 126 ± 12 135 ± 5 33 ± 3 46 ± 4 43 ± 3 77.3 ± 8.2 57.5 ± 7.9 77.2 ± 5.2 24 h 38.8 ± 0.1 39.3 ± 0.2 38.7 ± 0.2 136 ± 11 136 ± 15 120 ± 10 52 ± 11 43 ± 6 46 ± 10 192.3 ± 38.2 191.0* ± 25.3 159.5 ± 16.8 48 h 38.5 ± 0.2 39.7* ± 0.3 39.7* ± 0.3 128 ± 14 138 ± 14 141 ± 8 38 ± 8 41 ± 5 33 ± 1 196.0 ± 28.7 240.3* ± 59.8 130.7 ± 22.3 Values are presented as mean ± SEM and * indicates a significant (P < .05) difference from the BL value. S-FR (n = 3), LV (n = 6), MB (n = 6). BL, baseline; LV, limited volume; MB, modified brooke. Open in new tab Circulating glucose (a potential indicator of cortisol-induced hyperglycemia) was variable between timepoints due to feeding schedule. Collectively in all animals, plasma glucose (Table 1) was elevated by 24 hours (180.94 ± 10.73 mg/dl; P < .001) and 48 hours (189.00 ± 31.85 mg/dl; P < .001) when compared with BL levels (70.67 ± 6.58 mg/dl). Importantly, all of these timepoints represent blood draws that occurred in the morning time after overnight fasting. Despite a trend for reduction toward BL levels in the MB group, this did not reach significant reduction. Cortisol Is Elevated With Burn Conversion of cholesterol to cortisol is regulated by expression of key steroidogenic enzymes in the adrenal gland (Figure 1A). Figure 1A depicts fold regulation of genes involved in cortisol synthesis relative to S-FR evaluated 48 hours following burn injury. A slight downregulation in cytochrome P450 family 11 subfamily A member 1 (CYP11a1), Cytochrome P450 Family 17 Subfamily A Member 1 (CYP17a1), 3β-Hydroxysteroid dehydrogenase (3β-HSD), Cytochrome P450 Family 21 Subfamily A Member 2 (CYP21a2) was apparent after burn, but only Cyp17a1 approached statistical significance (P ≤ .08). It is important to note that transcriptional changes can happen on the order of hours, and 2 days may not be optimal to detect differences in this model. Figure 1. Open in new tabDownload slide Diagram of cholesterol to cortisol synthesis and the steroidogenic enzymes (gray) required for conversion (A). Fold Regulation (blue LV and green MB) of steroidogenic enzymes cytochrome P450 family 11 subfamily A member 1 (CYP11a1), Cytochrome P450 Family 17 Subfamily A Member 1 (CYP17a1), 3β-Hydroxysteroid dehydrogenase (3β-HSD), Cytochrome P450 Family 21 Subfamily A Member 2 (CYP21a2). Plasma cholesterol (B) and urinary cortisol (C) are quantified at baseline (BL), 6, 12, 24, and 48 hours following burn injury. Values are presented as mean ± SEM and * indicates a significant (P < .05) difference from the BL value and # indicates a difference (P < .05) between treatment(s) at the indicated timepoint. S-FR (n = 3), LV (n = 6), MB (n = 6). LV, limited volume; MB, modified brooke. Figure 1. Open in new tabDownload slide Diagram of cholesterol to cortisol synthesis and the steroidogenic enzymes (gray) required for conversion (A). Fold Regulation (blue LV and green MB) of steroidogenic enzymes cytochrome P450 family 11 subfamily A member 1 (CYP11a1), Cytochrome P450 Family 17 Subfamily A Member 1 (CYP17a1), 3β-Hydroxysteroid dehydrogenase (3β-HSD), Cytochrome P450 Family 21 Subfamily A Member 2 (CYP21a2). Plasma cholesterol (B) and urinary cortisol (C) are quantified at baseline (BL), 6, 12, 24, and 48 hours following burn injury. Values are presented as mean ± SEM and * indicates a significant (P < .05) difference from the BL value and # indicates a difference (P < .05) between treatment(s) at the indicated timepoint. S-FR (n = 3), LV (n = 6), MB (n = 6). LV, limited volume; MB, modified brooke. Cholesterol levels (Figure 1B) in all animals remained constant relative to BL levels until 48 hours when S-FR animals tended to display greater values (P = .065) relative to their BL values (92.33 ± 3.48 vs 66.33 ± 1.86 mg/dl, respectively) and were greater than MB animal values (58.67 ± 5.02 mg/dl; P = .01). Surgical preparations performed prior to burn (ie, animal cage transfer, sedation, surgical cut down, catheter line placement) proved stressful as BL plasma cortisol values in all animals were elevated (59.95 ± 8.87 ng/ml) and reduced at 48 hours in S-FR (20.54 ± 3.07 ng/ml), LV (21.46 ± 8.23 ng/ml), and MB (35.44 ± 6.87 ng/ml). Alternatively, urinary cortisol (Figure 1C) at BL was relatively low in all animals (85 ± 11 ng/mg creatinine), and therefore used for subsequent measurements. Six hours following the injury cortisol increased in all groups including SF-R (157 ± 19 ng/mg creatinine), LV (238 ± 44 ng/mg creatinine), and MB (211 ± 57 ng/mg creatinine) animals. At 24 hours in LV and MB animals (158 ± 41 and 139 ± 302 ng/mg creatinine, respectively) urinary cortisol remained elevated, whereas urinary cortisol levels in S-FR animals fell to near BL (64 ± 8 ng/mg creatinine). By 48 hours, levels slightly decreased in LV (122 ± 33 ng/mg creatinine) and MB (133 ± 17 ng/mg creatinine) and were statistically similar to their BL levels However, cortisol levels remained greater in MB (P = .0013) and LV (P = .03) when compared with S-FR (40 ± 5 ng/ml). CT Imaging of Adrenal Glands Did Not Detect Changes in Volume At necropsy, no gross abnormalities were observed of the collected adrenal gland in any animals as they all were intact, of similar shape and size, and bright red in color (Figure 2A). To assess adrenal gland integrity, CT angiographies were taken at BL and 48 hours (Figure 2B). No significant differences were detected at BL between treatments for adrenal gland volume (P = .97; Figure 2C) and perfusion (P = .35; Figure 2D). Volume tended (P = .12) to be greater in MB animals when compared to S-FR animals. Hounsfield units as a measure of perfusion was reduced (P = .009) to (94.7 ± 2.76 HU) 48 hours following BL (106.9 ± 3.98 HU) in all burned animals. Perfusion at 48 hours was greatest in S-FR (108.5 ± 6.7 HU; P = .03) when compared to LV (95.1 ± 3.6 HU) and MB (88.18 ± 3.2 HU; Figure 2D). Despite these observations, wet-to-dry adrenal gland weights were similar among groups (Figure 2E). Figure 2. Open in new tabDownload slide (A) Adrenal glands isolated immediately after euthanasia demonstrated no gross abnormalities between animals (representative picture shown is from MB group). (B) Computed tomography (CT) scanning was performed pre-injury and immediately prior to euthanasia (termination of experiment 48 hours; representative picture shown is from MB group). (C) Adrenal volume and Hounsfield units (D) were quantified at 48 hours. (E) Wet-to-dry values. Values are presented as mean ± SEM and * indicates a significant (P < .05) treatment difference. Figure 2. Open in new tabDownload slide (A) Adrenal glands isolated immediately after euthanasia demonstrated no gross abnormalities between animals (representative picture shown is from MB group). (B) Computed tomography (CT) scanning was performed pre-injury and immediately prior to euthanasia (termination of experiment 48 hours; representative picture shown is from MB group). (C) Adrenal volume and Hounsfield units (D) were quantified at 48 hours. (E) Wet-to-dry values. Values are presented as mean ± SEM and * indicates a significant (P < .05) treatment difference. Histopathology Showing Adrenal Hemorrhage and Apoptosis Representative images for adrenal sections stained for H&E are shown in Figure 3, which illustrate varying levels of hemorrhage in all animals and amongst different locations within the adrenal (Figure 3A–F). Severity of hemorrhage was greatest in the glomerulosa (P = .004) of MB animals when compared with S-FR or LV (P = .08; Figure 3G). While similar trends were seen in other locations, no significant differences were detected within the fasiculata/reticularis, or the medulla. To quantify the level of apoptosis-mediated cellular death within the cortex and medulla of the adrenal gland, fixed sections were stained with TUNEL and DAPI.27 Intensity of TUNEL stain on adrenal cortex sections was greater in the MB group (P < .01) and LV group (P = .14) when compared to S-FR (Figure 4C). Within the medulla, there was also detectable TUNEL staining in all animals, with a tendency (Figure 4D) for greater expression in the MB group (P = .14). The apoptosis-mediating molecule c-Jun N-terminal kinase (JNK) (Figure 4E) was quantified in total adrenal homogenates and levels were significantly greater in MB (P = .03), and moderately greater in LV (P =.10) animals when compared with SF-R. Figure 3. Open in new tabDownload slide Representative hematoxylin and eosin (H&E) staining of the adrenal in S-FR (A and D), LV (B and E), and MB (C and F) animals. Images demonstrate hemorrhaging which are graphically represented as mean ± SEM of % surface area in the glomerulosa (G), fasiculata/reticularis (H), and medulla (I). S-FR (n = 3), LV (n = 4), MB (n = 4). Scale bars represent 1 mm for A–C, and 200 µm for D–F. An * indicates a significant (P < .05) treatment difference. LV, limited volume; MB, modified brooke. Figure 3. Open in new tabDownload slide Representative hematoxylin and eosin (H&E) staining of the adrenal in S-FR (A and D), LV (B and E), and MB (C and F) animals. Images demonstrate hemorrhaging which are graphically represented as mean ± SEM of % surface area in the glomerulosa (G), fasiculata/reticularis (H), and medulla (I). S-FR (n = 3), LV (n = 4), MB (n = 4). Scale bars represent 1 mm for A–C, and 200 µm for D–F. An * indicates a significant (P < .05) treatment difference. LV, limited volume; MB, modified brooke. Figure 4. Open in new tabDownload slide Representative adrenal TUNEL- and DAPI-stained adrenal cortex (A) and medulla (B) sections shows staining intensity and thus apoptosis between S-FR, LV, and MOB treatments. (C) Quantification of color density reveals significantly greater apoptosis in the cortex and medulla (D) of MB-treated swine. Total JNK protein expression in adrenals was higher in the MB when compared to the S-FR and LV groups (E). Values are presented as mean ± SEM. S-FR (n = 3), LV (n = 4), MB (n = 4) and * indicates a significant (P < .05) treatment difference. Scale bars for all images represent 100 µm. JNK, c-Jun N-terminal kinase; LV, limited volume; MB, modified brooke; TUNEL, Terminal deoxynucleotidyl transferase dUTP nick-end labeling. Figure 4. Open in new tabDownload slide Representative adrenal TUNEL- and DAPI-stained adrenal cortex (A) and medulla (B) sections shows staining intensity and thus apoptosis between S-FR, LV, and MOB treatments. (C) Quantification of color density reveals significantly greater apoptosis in the cortex and medulla (D) of MB-treated swine. Total JNK protein expression in adrenals was higher in the MB when compared to the S-FR and LV groups (E). Values are presented as mean ± SEM. S-FR (n = 3), LV (n = 4), MB (n = 4) and * indicates a significant (P < .05) treatment difference. Scale bars for all images represent 100 µm. JNK, c-Jun N-terminal kinase; LV, limited volume; MB, modified brooke; TUNEL, Terminal deoxynucleotidyl transferase dUTP nick-end labeling. Cytokines Are Elevated With Low Volume IV Resuscitation Proinflammatory and anti-inflammatory cytokine levels in adrenal lysates were quantified (Figure 5). Within the tissue, levels of anti-inflammatory cytokines IL-10 (P = .03) and IL1ra (P < .05) were greater in LV when compared with MB animals. IL-4 tended to be greater in S-FR animals when compared to MB (P = .08). However, proinflammatory cytokines IL-12 and IL-1β were also greatest in LV animals when compared to S-FR (P = .04 and P = .13, respectively) and MB (P = .02 and P = .13, respectively) animals. Alternatively, levels of IL-6 tended to be greater in MB (P = .02) and LV (P = .10) animals when compared with S-FR. Figure 5. Open in new tabDownload slide Cytokines IL-4 (A), IL1ra (B), IL-6 (C), IL-10 (D), IL-12 (E), and IL-1β (F) were quantified in adrenal tissue and values are presented as mean ± SEM of the observed concentration and * indicates a significant (P < .05) treatment difference. S-FR (n = 3), LV (n = 4), MB (n = 5). LV, limited volume; MB, modified brooke. Figure 5. Open in new tabDownload slide Cytokines IL-4 (A), IL1ra (B), IL-6 (C), IL-10 (D), IL-12 (E), and IL-1β (F) were quantified in adrenal tissue and values are presented as mean ± SEM of the observed concentration and * indicates a significant (P < .05) treatment difference. S-FR (n = 3), LV (n = 4), MB (n = 5). LV, limited volume; MB, modified brooke. DISCUSSION Patients with burns experience fluctuations in plasma cortisol that are proportional to the size of the burn.4 Typically this results in greater circulating cortisol concentrations and increased cortisol production lasting several days after burn.4,28 With all of the pathological stressors that take place after burn injury, the role of the adrenal gland is crucial for patient outcome. For example, Curling’s ulcer, an acute ulceration of the stomach or duodenum in some patients with burns, may be ultimately brought on by elevated stress and cortisol.29,30 Although changes in cortisol are a normal adaptation mechanism to stress and injury, if left unregulated for a period of time, it can lead to detrimental outcomes. Many researchers have demonstrated cortisol fluctuations following burn injury, and while mechanistic examination of the adrenal response following burn is possible with animal models, this remains largely unstudied. The current study utilizes a porcine 40% TBSA burn model to report that volumes of IV resuscitation with LR can affect the response of the adrenal following burn injury. Specifically, although greater volumes of fluids exacerbated hemorrhaging and apoptosis postburn within the adrenal gland, this did not cause an increased inflammatory profile. Other salient findings presented herein demonstrate that urinary cortisol is increased after burn injury with no major effect of IV fluid volume, indicating this may represent a noninvasive marker of the transition from the “ebb” (shock) phase to the “flow” (protein and fat turnover for energy maintenance) phase of burn pathogenesis.31 While the temporal aspect of urinary cortisol concentrations needs further delineation, this type of information could inform treatments strategy such as timing of surgical intervention. In this study burned pigs had greater cortisol levels than S-FR, but they recovered to near BL levels by 48 hours. Interestingly, control animals that were not burned (S-FR) displayed more stressed behavior and therefore required midazolam. The reason for this behavior is likely from S-FR animals not being thermally injured and therefore are more aware of the metabolic cage environment (ie, the burn injury itself caused mild sedation and lethargy). Despite this, S-FR animals’ cortisol levels were not as consistently elevated as the burned animals. Patients with burns >20% TBSA have a 40% reduced cholesterol that correlates to greater infection rates and length of stay, as well as a prolonged reduction in cortisol.32 Total cholesterol was measured to ascertain whether burn injury similarly depletes levels in this swine model, and if this is affected by changes in cortisol production due to the resuscitation regimen. Interestingly, we found no such relationship herein, and key steroidogenic enzymes responsible for the production of cortisol were actually lower than sham animals, albeit nonsignificantly. As mentioned earlier, the expression of these enzymes may have risen sharply after burn injury, and had already decreased to BL values by 48 hours. Chronic stress has been reported to increase adrenal gland weight in rats; therefore, a CT scan at BL and 48 hours was performed to detect changes in adrenal gland volume and perfusion.33 While minor changes in adrenal volume were detected, significant changes may be realized with an increase in animal number. On the contrary, burn injury reduced perfusion of the adrenal (HU; Figure 2) as S-FR animals displayed similar levels to BL that were significantly higher than both groups at 48 hours. Postmortem evaluation of adrenal glands provided more insight into the degree of burn-induced damage. Hemorrhage was not macroscopically observed in the adrenals during organ harvest at necropsy, as has been reported in some patients with burns.10,11 However, histological observation demonstrated hemorrhaging and cellular death postburn. Furthermore, animals receiving larger fluid volumes displayed greater hemorrhaging and apoptosis in the adrenals when compared to S-FR animals. Moreover, adrenal hemorrhaging in the glomerulosa and TUNEL expression measured histologically were positively correlated with each other (r = .77, P = .006). While the implications of this relationship is unclear, JNK proved to be a key signaling molecule mediating apoptosis in this model, as expression of JNK also positively correlated to TUNEL staining within both the cortex (R = .71, P = .01) and medulla (R = .62, P = .04). The drawbacks or benefits of apoptosis within the adrenal postburn injury warrants further investigation, as many different molecular targets exist that could be leveraged for therapeutic purposes. Given the importance of IV fluids in burn injury, the above TUNEL stain finding may seem counter-intuitive. Indeed, limiting IV fluids in the burn patient can lead to deleterious consequences as proven by improved outcomes using large volumes in the last several decades. As such, the increased apoptosis may represent a protective mechanism in which programmed death of compromised cells potentiate recovery of the HPA axis. We have recently shown that limiting volumes of enteral fluids in this model exacerbates AKI.34 Moreover, a recent study suggested that limited volumes of IV fluids (although still within the range of 2–4 ml/kg/%TBSA) may predispose patients to AKI.35 In our study, the low volume group did show a low urine output and elevated creatinine suggesting that volumes of IV fluids below the standard of care are not optimal. While limited IV fluids may be a problem in austere environments and mass casualty scenarios, the nationwide shortage of IV fluids due to the recent hurricanes in Puerto Rico may also limit fluid administration. In this light the use of alternative IV fluids or oral resuscitation for the treatment of burn treatment warrants further investigation. While previous investigators identified that cholesterol was inversely correlated to plasma IL-6,32 cytokine levels in adrenal tissue after burn injury has not been reported to date. In our model, protein levels of IL-6 in adrenal tissue were greatest in the MB group when compared to the other groups. These results align with several others demonstrating IL-6 levels correlate with burn injury and severity.36,37 Additionally, adrenal IL1ra and IL-10 levels were greatest in the LV relative to the MB group. While these cytokines are also implicated in burns,38,39 this data would seem to indicate that a conservative fluid regimen increases levels of anti-inflammatory cytokines within adrenal tissue. However, this phenomenon was nonspecific, as levels of proinflammatory cytokine IL-12 was also higher with limited fluids. In this regard, greater volumes of IV fluids given in our MB group reduced levels of most cytokines, with the exception of IL-6. However, no differences were detected in wet-to-dry ratios, thus eliminating the possibility of simple dilution of these proteins. Contrary to our hypothesis, the apoptosis and hemorrhaging mentioned above did not result in a locally exacerbated inflammation. There are limitations of this study worth mentioning. Swine are highly intelligent animals and cognitive stress associated with abrupt changes in their normal day to day activities (eg, surgery preparation)40 prevented normal, unstressed plasma cortisol measurements. Further investigation on cortisol levels could utilize urinary or salivary measurements as a nonstress-inducing method of obtaining measurements. Although this study was not intended to analyze animal behavior after burn, nonburned animals displayed behavior of being more stressed from placement into the metabolic cage, which necessitated the exclusion of one animal from the S-FR group. This negatively affected detection of significance levels, and there are several examples of data presented herein that approached statistical significance. Along the same lines, the greater volume of fluids administered in this study (ie, MB) represents a moderate level of IV fluids that is at the lower end of the standard of care spectrum. IV fluid volumes according to the Parkland formula (4 ml/kg/%TBSA/d, double that given in this study) would have likely exacerbated the volume-associated findings in hemorrhaging and apoptosis, we chose to study more limited volumes for application to resource poor setting (eg, prolonged field care, wilderness medicine, mass causality events). Other limitations are commonly associated with swine, and include the lack of appropriate molecular tools (eg, antibodies/primers) and high cost precluding the analysis of tissue protein and gene expression over time. Nevertheless, we believe that our model can offer insights into the early stress response, the mechanisms associated with the cortisol response, and the role of the adrenal gland postburn injury. CONCLUSION Patients with burns experience fluctuations in cortisol production that are normal to help cope with the degree of injury. This study demonstrates in a controlled animal study that urinary cortisol levels may provide a noninvasive diagnostic tool delineating the transition from ebb to flow phases of burn injury. Moreover, cellular death (particularly in the adrenal medulla) is exacerbated with greater volumes of resuscitation fluid which is also associated with adrenal hemorrhaging. This does not, however, lead to inflammation as both pro- and anti-inflammatory cytokines in adrenal tissue were nonspecifically regulated. The findings in this study highlight the acute effects of burn on the adrenal gland and suggest further mechanistic studies are warranted. Conflict of interest statement. None declared. This study was supported by Congressionally Directed Medical Research Program Award# W81XWH-16-2-0041. The opinions or assertions contained herein are the private views of the author and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense. Supplemental digital content is available for this article. DirectURL citations appear in the printed text and are provided in theHTML and PDF versions of this article on the journal’s Web site. REFERENCES 1. Burmeister DM , Gómez BI, Dubick MA. Molecular mechanisms of trauma-induced acute kidney injury: inflammatory and metabolic insights from animal models . Biochim Biophys Acta 2017 ; 1863 ( 10 Pt B ): 2661 – 71 . Google Scholar Crossref Search ADS PubMed WorldCat 2. Atiyeh BS , Dibo SA, Ibrahim AE, Zgheib ER. Acute burn resuscitation and fluid creep: it is time for colloid rehabilitation . Ann Burns Fire Disasters 2012 ; 25 : 59 – 65 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 3. Kollias S , Stampolidis N, Kourakos Pet al. Abdominal compartment syndrome (ACS) in a severely burned patient . Ann Burns Fire Disasters 2015 ; 28 : 5 – 8 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 4. Vaughan GM , Becker RA, Allen JP, Goodwin CW Jr, Pruitt BA Jr, Mason AD Jr. Cortisol and corticotrophin in burned patients . J Trauma 1982 ; 22 : 263 – 73 . Google Scholar Crossref Search ADS PubMed WorldCat 5. Bergquist M , Huss F, Fredén Fet al. Altered adrenal and gonadal steroids biosynthesis in patients with burn injury . Clin Mass Spectrom 2016 ; 1 : 19 – 26 . Google Scholar Crossref Search ADS WorldCat 6. Wise L , Margraf HW, Ballinger WF. Adrenal cortical function in severe burns . Arch Surg 1972 ; 105 : 213 – 20 . Google Scholar Crossref Search ADS PubMed WorldCat 7. Khansari DN , Murgo AJ, Faith RE. Effects of stress on the immune system . Immunol Today 1990 ; 11 : 170 – 5 . Google Scholar Crossref Search ADS PubMed WorldCat 8. Lephart ED , Baxter CR, Parker CR , Jr. Effect of burn trauma on adrenal and testicular steroid hormone production . J Clin Endocrinol Metab 1987 ; 64 : 842 – 8 . Google Scholar Crossref Search ADS PubMed WorldCat 9. Sheridan RL , Ryan CM, Tompkins RG. Acute adrenal insufficiency in the burn intensive care unit . Burns 1993 ; 19 : 63 – 6 . Google Scholar Crossref Search ADS PubMed WorldCat 10. Vijay Kumar AG , Kumar U, Shivaramu MG. Adrenal haemorrhages and burns – an autopsy study . J Forensic Res 2012 ; 3 : 162 – 4 . Google Scholar OpenURL Placeholder Text WorldCat 11. Weiskotten HG . Fatal superficial burns and the suprarenals: note on the occurrence of suprarenal lesions in uncomplicated fatal cases of extensive superficial burns . JAMA 1917 ; LXIX : 776 . Google Scholar Crossref Search ADS WorldCat 12. Xarli VP , Steele AA, Davis PJ, Buescher ES, Rios CN, Garcia-Bunuel R. Adrenal hemorrhage in the adult . Medicine (Baltimore) 1978 ; 57 : 211 – 21 . Google Scholar Crossref Search ADS PubMed WorldCat 13. Nácul FE , Jardim A, MacCord F, Penido C, Gomes MV. Hemodynamic instability secondary to adrenal insufficiency in a major burn patient . Burns 2002 ; 28 : 270 – 72 . Google Scholar Crossref Search ADS PubMed WorldCat 14. Neary N , Nieman L. Adrenal insufficiency: etiology, diagnosis and treatment . Curr Opin Endocrinol Diabetes Obes 2010 ; 17 : 217 – 23 . Google Scholar Crossref Search ADS PubMed WorldCat 15. Murphy JF , Purdue GF, Hunt JL. Acute adrenal insufficiency in the patient with burns . J Burn Care Rehabil 1993 ; 14 ( 2 Pt 1 ): 155 – 7 . Google Scholar Crossref Search ADS PubMed WorldCat 16. Chao T , Porter C, Herndon DNet al. Propranolol and oxandrolone therapy accelerated muscle recovery in burned children . Med Sci Sports Exerc 2018 ; 50 : 427 – 35 . Google Scholar Crossref Search ADS PubMed WorldCat 17. Fang CH , James HJ, Ogle C, Fischer JE, Hasselgren PO. Influence of burn injury on protein metabolism in different types of skeletal muscle and the role of glucocorticoids . J Am Coll Surg 1995 ; 180 : 33 – 42 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 18. Lang CH , Silvis C, Nystrom G, Frost RA. Regulation of myostatin by glucocorticoids after thermal injury . FASEB J 2001 ; 15 : 1807 – 9 . Google Scholar Crossref Search ADS PubMed WorldCat 19. Fukuzuka K , Edwards CK 3rd, Clare-Salzler M, Copeland EM 3rd, Moldawer LL, Mozingo DW. Glucocorticoid-induced, caspase-dependent organ apoptosis early after burn injury . Am J Physiol Regul Integr Comp Physiol 2000 ; 278 : R1005 – 18 . Google Scholar Crossref Search ADS PubMed WorldCat 20. Burmeister DM , McIntyre MK, Baker BAet al. Impact of isolated burns on major organs: a large animal model characterized . Shock 2016 ; 46 ( 3 Suppl 1 ): 137 – 47 . Google Scholar Crossref Search ADS PubMed WorldCat 21. Burmeister DM , McIntyre MK, Beely Bet al. A model of recovery from inhalation injury and cutaneous burn in ambulatory swine . Burns 2017 ; 43 : 1295 – 305 . Google Scholar Crossref Search ADS PubMed WorldCat 22. Burmeister DM , Ponticorvo A, Yang Bet al. Utility of spatial frequency domain imaging (SFDI) and laser speckle imaging (LSI) to non-invasively diagnose burn depth in a porcine model . Burns 2015 ; 41 : 1242 – 52 . Google Scholar Crossref Search ADS PubMed WorldCat 23. Kelley KW , Curtis SE, Marzan GT, Karara HM, Anderson CR. Body surface area of female swine . J Anim Sci 1973 ; 36 : 927 – 30 . Google Scholar Crossref Search ADS PubMed WorldCat 24. Al-Shaibi A. Investigating impacts of altered central metabolism on ovarian function . Graduate Theses and Dissertations ; 2016 . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC 25. Wojciechowicz B , Kotwica G, Kolakowska J, Franczak A. The activity and localization of 3β-hydroxysteroid dehydrogenase/Δ(5)-Δ(4) isomerase and release of androstenedione and progesterone by uterine tissues during early pregnancy and the estrous cycle in pigs . J Reprod Dev 2013 ; 59 : 49 – 58 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 26. Kaminska B , Czerwinska J, Wojciechowicz B, Nynca A, Ciereszko R. Genistein and daidzein affect in vitro steroidogenesis but not gene expression of steroidogenic enzymes in adrenals of pigs . J Physiol Pharmacol 2014 ; 65 : 127 – 33 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 27. Loo DT . In situ detection of apoptosis by the TUNEL assay: an overview of techniques . Methods Mol Biol 2011 ; 682 : 3 – 13 . Google Scholar Crossref Search ADS PubMed WorldCat 28. Hume DM , Nelson DH, Miller DW. Blood and urinary 17-hydroxycorticosteroids in patients with severe burns . Ann Surg 1956 ; 143 : 316 – 29 . Google Scholar Crossref Search ADS PubMed WorldCat 29. Pruitt BA Jr, Foley FD, Moncrief JA. Curling’s ulcer: a clinical-pathology study of 323 cases . Ann Surg 1970 ; 172 : 523 – 39 . Google Scholar Crossref Search ADS PubMed WorldCat 30. Shenkin HA , Feldman W. Plasma cortisol binding globulin levels in gastro-intestinal bleeding of stress origin . J Clin Endocrinol Metab 1968 ; 28 : 15 – 18 . Google Scholar Crossref Search ADS PubMed WorldCat 31. Tredget EE , Yu YM. The metabolic effects of thermal injury . World J Surg 1992 ; 16 : 68 – 79 . Google Scholar Crossref Search ADS PubMed WorldCat 32. Vanni HE , Gordon BR, Levine DMet al. Cholesterol and interleukin-6 concentrations relate to outcomes in burn-injured patients . J Burn Care Rehabil 2003 ; 24 : 133 – 41 . Google Scholar Crossref Search ADS PubMed WorldCat 33. Ulrich-Lai YM , Figueiredo HF, Ostrander MM, Choi DC, Engeland WC, Herman JP. Chronic stress induces adrenal hyperplasia and hypertrophy in a subregion-specific manner . Am J Physiol Endocrinol Metab 2006 ; 291 : E965 – 73 . Google Scholar Crossref Search ADS PubMed WorldCat 34. Gómez BI , McIntyre MK, Gurney JM, Chung KK, Cancio LC, Dubick MA, Burmeister DM. Enteral resuscitation with oral rehydration solution to reduce acute kidney injury in burn victims: evidence from a porcine model . PLoS One 2018 ; 13 ( 5 ): e0195615 . doi:10.1371/journal.pone.0195615 Google Scholar Crossref Search ADS PubMed WorldCat 35. Mason SA , Nathens AB, Jeschke MG. “ Hold the pendulum: rates of acute kidney injury are increased in patients who receive resuscitation volumes less than predicted by the parkland equation ”. Ann Surg 2017 ; 266 : e108 . Google Scholar Crossref Search ADS PubMed WorldCat 36. Hager S , Foldenauer AC, Rennekampff HOet al. Interleukin-6 serum levels correlate with severity of burn injury but not with gender . J Burn Care Res 2017 ; 39 ( 3 ): 379 – 86 . Google Scholar OpenURL Placeholder Text WorldCat 37. Kowal-Vern A , Walenga JM, Hoppensteadt D, Sharp-Pucci M, Gamelli RL. Interleukin-2 and interleukin-6 in relation to burn wound size in the acute phase of thermal injury . J Am Coll Surg 1994 ; 178 : 357 – 62 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 38. Endo S , Inada K, Yamada Yet al. Plasma levels of interleukin-1 receptor antagonist (IL-1ra) and severity of illness in patients with burns . J Med 1996 ; 27 : 57 – 71 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 39. Pileri D , Accardo Palombo A, D’Amelio Let al. Concentrations of cytokines IL-6 and IL-10 in plasma of burn patients: their relationship to sepsis and outcome . Ann Burns Fire Disasters 2008 ; 21 : 182 – 5 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 40. Smith AC , Swindle MM. Preparation of swine for the laboratory . ILAR J 2006 ; 47 : 358 – 63 . Google Scholar Crossref Search ADS PubMed WorldCat Published by Oxford University Press on behalf of the American Burn Association 2018. This work is written by (a) US Government employee(s) and is in the public domain in the US. Published by Oxford University Press on behalf of the American Burn Association 2018.
TI - Effect of Intravenous Fluid Volumes on the Adrenal Glucocorticoid Response After Burn Injury in Swine
JF - Journal of Burn Care & Research
DO - 10.1093/jbcr/iry024
DA - 2018-08-17
UR - https://www.deepdyve.com/lp/oxford-university-press/effect-of-intravenous-fluid-volumes-on-the-adrenal-glucocorticoid-x05t4Qwww5
SP - 652
EP - 660
VL - 39
IS - 5
DP - DeepDyve
ER -